Glutamate-induced excitotoxicity is increasingly recognized as the trigger for swelling, retraction, and delayed degeneration of auditory nerve fibers (ANFs) following moderate overexposure to sound; however, little is known about the underlying mechanisms. This excitotoxicity seems to involve glutamate receptors, and research in other systems has indicated the crucial role of postsynaptic intracellular Ca2+ in mediating the excitotoxicity that produces slow neurodegeneration. Postsynaptic Ca2+ can also mediate homeostatic plasticity. It is still unknown if or how Ca2+ signals link excitotoxicity to neurodegeneration or protection in ANFs. Although all ANF terminals express glutamate receptors, they differ in susceptibility to noise-induced synaptopathy and degeneration. The roles of glutamate receptor subunits in the inner ear deserve attention because glutamate-induced Ca2+ influx through receptors depends upon subunit composition. The project encompasses studies of activity-dependent synaptic plasticity in the cochlea because our long- term goal is to identify mechanisms of synaptic damage and repair that can be manipulated to prevent or rapidly reverse damage before the onset of neurodegeneration. We have already demonstrated that ANF terminals differ from each other in their complements of AMPA-type glutamate receptor subunits. We hypothesize that heterogeneity of glutamate receptor subunit expression among ANF terminals is a crucial determinant of susceptibility to noise-induced damage. Thus, we are studying noise-activated changes in subunit composition. We are comparing receptor subunit composition with presynaptic molecular anatomy and reconstructing synapse position on the inner hair cell (IHC) to compare along the modiolar-pillar and orthogonal axes. We are using genetically modified mice to manipulate glutamatergic activity. We previously employed superresolution STED microscopy to measure synaptic structures at 50 nm resolution in 2D. We now implement, for the first time in the organ of Corti, 3D superresolution STORM microscopy at 20 nm resolution. We are now able to measure the intrasynaptic organization of AMPA receptor subunits with subunit-specific antibodies to GluA2, GluA3, and GluA4. Anatomical measurements will be complemented with functional recordings. In prior work with the patch- clamp technique we made the first measurements of ANF excitability with direct current injection into ANF terminals. Here, differences in firing behavior will be compared with synaptic structure by filling recorded neurons with dye, followed by fixation and immunohistochemistry. We are implementing Ca2+ imaging in ANFs for the first time, which allows for less invasive, simultaneous observation of activity across fibers. We will use Ca2+ imaging to test for functional routes of Ca2+ entry pharmacologically. Understanding how ANF diversity is shaped by glutamate receptor subunits and postsynaptic Ca2+ will deliver new perspectives on questions of clinical hearing loss as well as the basic mechanisms underlying this unique synapse.